Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-EIF4EBP1 knockout cells (red line) stained with ab32024. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32024, 0.1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-EIF4EBP1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min) , permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32024).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

This data was developed using ab32024, the same antibody clone in a different buffer formulation. Intracellular Flow Cytometry analysis of HEK-293 (Human embryonic kidney epithelial cell) cells labeling eIF4EBP1 with purified ab32024 at 1/20 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling eIF4EBP1 with Purified ab32024 at 1/500 dilution (5 µg/ml). Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) at 1/1000 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32024).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

This data was developed using ab32024, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon cancer tissue sections labeling eIF4EBP1 with purified ab32024 at 1/200 dilution (0.53 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

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Immunoprecipitation - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

This data was developed using ab32024, the same antibody clone in a different buffer formulation.
Purified ab32024 at 1/20 dilution (2μg) immunoprecipitating eIF4EBP1 in HEK-293 whole cell lysate.
Lane 1 (input) : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab32024 + HEK-293 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32024 in HEK-293 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 15-20 kDa

All lanes:

Immunoprecipitation - Anti-eIF4EBP1 antibody [Y329] (<a href='/en-us/products/primary-antibodies/eif4ebp1-antibody-y329-ab32024'>ab32024</a>)

Predicted band size: 13 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

This data was developed using ab32024, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue sections labeling eIF4EBP1 with purified ab32024 at 1/200 dilution (0.53 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

This data was developed using ab32024, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue sections labeling eIF4EBP1 with purified ab32024 at 1/200 dilution (0.53 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

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Western blot - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

This data was developed using ab32024, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-eIF4EBP1 antibody [Y329] (<a href='/en-us/products/primary-antibodies/eif4ebp1-antibody-y329-ab32024'>ab32024</a>) at 1/2000 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 3:

Mouse skeletal muscle lysate at 20 µg

Lane 4:

Rat skeletal muscle lysate at 20 µg

Lane 5:

L6 (Rat skeletal muscle myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 13 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-eIF4EBP1 antibody [Y329] - BSA and Azide free (AB173370)

This data was developed using the same antibody clone in a different buffer formulation (ab32024).

Lanes 1-2 : Merged signal (red and green). Green - ab32024 observed at 13 kDa. Red - loading control ab8245 observed at 37 kDa.

ab32024 Anti-eIF4EBP1 antibody [Y329] was shown to specifically react with eIF4EBP1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264784 (knockout cell lysate ab257146) was used. Wild-type and eIF4EBP1 knockout samples were subjected to SDS-PAGE. ab32024 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 Dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-eIF4EBP1 antibody [Y329] (<a href='/en-us/products/primary-antibodies/eif4ebp1-antibody-y329-ab32024'>ab32024</a>) at 1/5000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human EIF4EBP1 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-eif4ebp1-knockout-hela-cell-lysate-ab257146'>ab257146</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 13 kDa

Observed band size: 13 kDa,37 kDa

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